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6 Cell-Penetrating Peptides: Processes and Applications
FIGURE 1.1 Cellular internalization of the Tat peptide. The primary sequence of the Tat
peptide is indicated in the three-letter code. Left panel: fluorescence microscopy study of the
internalization of the fluoresceinated Tat peptide: U2OS cells were incubated for 30 min at
37°C with 1 µM of fluorescein-labeled Tat peptide. Peptide shows a nuclear localization with
a nucleolar concentration as previously shown in HeLa cells. 23 Right panel: FACS analysis
of the uptake of a rhodamine-labeled Tat peptide in HeLa cells. Extracellular concentrations
of peptide were as indicated on the scheme. Peptide internalization could be easily detected
from 100 nM concentration. Signal appeared to saturate above 1 µM concentration.
have been monitored by various protocols in order to avoid potential artefacts. In
most cases, the Tat-derived peptides were conjugated through an additional Cterminal
cysteinyl residue to fluorescein and fluorescence distribution was analyzed
on formaldehyde fixed cells or in living cells, according to published protocols. 23
Alternatively, the intracellular distribution of Tat has been analyzed by indirect
immunofluorescence with a monoclonal antibody specific to the Tat basic domain.
Since uptake of the Antennapedia third helix had been ascribed to an amphipathic
helix, we then compared the cellular internalization of a series of Tat-derived peptides
carrying deletions. The importance of the basic region was confirmed by the lack
of uptake of a peptide including the full α-helix domain but carrying deletions in
the C-terminal cluster of basic amino acids (peptides 38–54). The 48–60 peptide
encompassing the basic amino acids stretch and lacking the potential α-helix was
the most efficiently internalized (Figure 1.1).
Interestingly, amino acids substitutions in the Antennapedia homeodomain also
ruled out the importance of the α-amphipathic helix configuration for cellular internalization.
17 The next issues to establish were whether this sequence could be
shortened without loss of internalization efficiency and whether its strong basic
character was essential. A series of progressive deletions and amino acids replacements
(Ala scan) were performed, as summarized in Figure 1.2.